Musculoskeletal diseases include a multitude of disorders that are prevalent in aging populations (Picavet and Hazes, Ann. Rheum. Dis. 62(7):644-650 (2003), Leveille, Curr. Opin. Rheumatol. 16(2):114-118 (2004), Harkness et al, Rheumatology (Oxford) 44(7):890-895 (2005)). Global population studies and World Health Organization statistics indicate that 10-50% of individuals suffer from musculoskeletal disorders and up to 3% will be classified as disabled due to their bone and joint conditions (Kean and Buchanan, Inflammopharmacology 13(4):343-370 (2005)). The most widely occurring forms of musculoskeletal disease are osteoporosis and osteoarthritis (OA). Globally, OA is estimated to affect 9.6% of men and 18% of women ≧60 years old (Woolf and Pfleger, Bull. World Health Organ. 81(9):646-656 (2003)). Osteoporosis affects as much as 30% of the post-menopausal women in the US, and 23% of women aged ≧50 in the UK (Woolf and Pfleger, Bull. World Health Organ. 81(9):646-656 (2003)). The growing physical and financial burden of these diseases has lead to the research and development of numerous musculoskeletal disease related biomarkers, many of which are fragments of long-lived proteins.
Cartilage is composed of two major protein constituents, collagen II and proteoglycan (Kuettner et al, Modern aspects of articular cartilage biochemistry, Cartilage Changes in Osteoarthritis, K. Brandt, Ciba Geigy, 3-11 (1990)). Proteoglycans, entrapped in the collagen network, make up ˜40% of the dry weight of cartilage. The proteoglycan component plays a crucial role in the structure of cartilage by endowing the tissue with its ability to reversibly absorb loads (Kuettner et al, Modern aspects of articular cartilage biochemistry, Cartilage Changes in Osteoarthritis, K. Brandt, Ciba Geigy, 3-11 (1990)), so-called compressive stiffness. Aggrecan is the major proteoglycan found in articular cartilage forming the protein component of the hydrated gel matrix (Cs-Szabo et al, Arthritis Rheum. 38(5):660-668 (1995), Knudson and Knudson, Semin. Cell Dev. Biol. 12(2):69-78 (2001), Kiani et al, Cell Res. 12(1):19-32 (2002)). Collagen II makes up ˜60% of the dry weight of cartilage and provides tensile stiffness and strength. Collagen architecture of normal articular cartilage consists of layers of flat ribbons parallel to the surface, vertical columns in the intermediate zone and a random meshwork in the deep zone (Hwang et al, J. Pathol. 167:425-433 (1992)). Cartilage, like bone, is in a continual state of resorption and formation.
A number of studies have found evidence for an enhanced synthesis of extracellular matrix components in OA (Lippiello et al, J. Clin. Invest. 593-600 (1977); Eyre et al, Biochem. J. 823-837 (1980); Mankin et al, J. Bone Joint Surg. 131-139 (1981)). By enhancing their anabolic or formation activity, chondrocytes attempt to repair the damaged matrix. Little is currently known about the capacity of different joints to repair damaged matrix or about the balance between degradation and repair for different joint sites.
More is known about the balance between degradation and repair at a protein level. One of the key factors in the etiology of musculoskeletal diseases is altered protein turnover. In altered protein turnover, the degradation and synthesis pathways in a tissue become dysregulated, resulting either in excess production or degradation. In OA, the physiological balance between extracellular matrix synthesis and degradation is altered in favor of degradation. This appears to be due to a cell-mediated upregulation of normal degradative processes in combination with the synthesis of poorly assembled matrix pools of molecules (Quinn et al, Ann. N.Y. Acad. Sci. 878:420-441 (1999)). Type II collagen is degraded by interstitial collagenases, a class of matrix metalloproteinases (MMPs) that possess the unique ability to cleave an intact triple helical collagen fibril (Eyre, Arthritis Res. 4(1):30-35 (2002), Eyre, Clin. Orthop. Relat. Res. (427 Suppl):S118-S122 (2004)). Damage to the fibrillar meshwork of cartilage is considered a serious and irreversible occurrence due to the slow rate of collagen turnover within cartilage. The catabolism of aggrecan is also mediated by a unique sub-group of metalloproteinases known as aggrecanases or ADAMTS (Nagase and Kashiwagi, Arthritis Res. Ther. 5(2):94-03 (2003)). The release of small molecular weight G1-bearing (globular fragment) species of aggrecan is commonly interpreted as a final stage in chondrocyte-mediated proteoglycan metabolism (Quinn et al, Ann. N.Y. Acad. Sci. 878:420-441 (1999)). Both type II collagen and aggrecan are degraded over the course of OA, are detectable in body fluids, and can be used as a measure of cartilage degradation.
Non-enzymatic modification of protein constitutes a byproduct of aging with potential biologic consequences. Non-enzymatic modifications that can occur in vivo include deamidation, oxidation, nitration, glycation, and racemization. Deamidation is believed to be a mechanism of amino acid damage and aging in numerous proteins and a variety of tissues (Robinson and Robinson, Molecular Clocks: Deamidation of Asparaginyl and Glutaminyl Residues in Peptides and Proteins, Cave Junction, Oreg., Althouse Press (2004)). Both asparagine (Asn) and glutamine (Gln) can be deamidated to form aspartate (Asp) and glutamate (Glu), respectively. Deamidation rates for Asn are usually faster than for Gln (Robinson and Robinson, Molecular Clocks: Deamidation of Asparaginyl and Glutaminyl Residues in Peptides and Proteins, Cave Junction, Oreg., Althouse Press (2004)). In fact, deamidation of Asn is thought to be possibly the single greatest means of producing protein damage under conditions of neutral pH (Aswad, Promega Notes Magazine 52:27 (1995)).
Deamidation rates are increased by increases in temperature, ionic strength, and pH (especially above pH 6). “Hot spots” for deamidation are predicted to exist as deamidation is known to vary based on factors such as steric hindrance and protein context (Robinson and Robinson, Molecular Clocks: Deamidation of Asparaginyl and Glutaminyl Residues in Peptides and Proteins, Cave Junction, Oreg., Althouse Press (2004), pp 97-116). For instance, particular peptide sequences are known to deamidate more readily than others, including GlyAsn, AsnGly and GlnGly (Robinson and Robinson, Molecular Clocks: Deamidation of Asparaginyl and Glutaminyl Residues in Peptides and Proteins, Cave Junction, Oreg., Althouse Press (2004)). Because joint tissues are at physiological pH or lower, this would tend to slow the overall rate of deamidation in joint tissues, particularly cartilage.
Protein turnover (anabolism and catabolism) rates can be used as a measure of tissue health in musculoskeletal disease. This is best exemplified by the use of type I collagen biomarkers for the monitoring of osteoporosis (Garnero, Mol. Diagn. Ther. 2008; 12(3):157-70 (2008)). Although a number of biomarkers have been associated with OA, none is yet approved for clinical use. Moreover, current biomarkers do not allow for the age of a given protein or protein fragment to be estimated or to be taken into account.
The present invention relates to methods of quantifying aged proteins and protein fragments (e.g., deamidated fragments of cartilage oligomeric matrix protein (COMP)) in body fluids, and thereby differentiating newly synthesized material from material derived by degradation of older resident molecules. By distinguishing older breakdown products from new protein fragments or total fragments (that is, fragments of all ages), the invention makes possible diagnostic, joint specific, and/or prognostic analyses of musculoskeletal diseases.